1. Field of the Invention
The present invention relates to a porous electrochemical electrode consisting of a semi-graphitized carbon monolith comprising a hierarchical porous network comprising interconnected macropores and micropores, the macropores of which contain an electroactive entity, to a process for preparing such an electrode, and to the use of such an electrode as a biosensor for detecting analytes in a liquid medium or for producing a biofuel cell.
2. Description of Related Art
A biosensor is an analytical tool or system consisting of an immobilized biological entity, known as a “ligand”, connected to a transducer which converts the biochemical signal into a quantifiable physical signal. According to the International Union of Pure and Applied Chemistry (IUPAC), a biosensor must be small and compact, have a reversible signal, give precise determinations (“onoff” reactions) and establish a real connection between the biological material and the transducer.
Electrochemical biosensors constitute a particular category of biosensors in which the ligand is immobilized on an electrode. In this case, the biochemical response to the addition of a substrate (analyte) is converted into an amplified and quantifiable electrical signal. Electrochemical biosensors may be amperometric, potentiometric, coulometric or conductimetric. Amperometric biosensors measure the current generated at a constant potential by an oxidoreduction reaction. Potentiometric biosensors measure the potential difference between an active electrode and a reference electrode. The ligands are biological compounds which make it possible to give the biosensor great specificity. The ligands most commonly used are enzymes and antibodies. However, whole cells, cell organelles, nucleic acids (DNA, RNA, oligonucleotides), antigens or alternatively receptors may also be used.
A biofuel cell is a system consisting of a cathode and an anode, to which bioelectrocatalysts of different nature (ligand) are attached, most often electroactive enzymes, the reactions of which with the substances present in the medium in which they are implanted generate an electric current which makes it possible to supply low-power equipment in varied fields such as the environment or health. By way of example of a biofuel cell, mention may in particular be made of the biofuel cells which use the chemical energy from the oxygen-glucose pair naturally present in physiological fluids to supply implanted medical devices intended, for example, to monitor blood glucose levels in diabetics. In this biofuel cell, a glucose oxidase is attached to the anode by means of a conductive polymer I and a bilirubin oxidase (BOD) is attached to the cathode by means of a conductive polymer II. When functioning, at the anode, the electrons are transferred from the glucose present in the physiological fluid to the glucose oxidase (GOx), and then from the GOx to the conductive polymer I and from the conductive polymer I to the anode. At the cathode, the electrons are transferred from the cathode to the conductive polymer II, then to the BOD and from the HOD to the oxygen present in the physiological fluid.
Irrespective of the application envisioned (electrochemical biosensor or biofuel cell), the ligand can be immobilized on the electrode by various, methods, such as adsorption, covalent coupling, encapsulation, etc.; the objective of any immobilization method is to retain the maximum activity of the biological ligand on the surface or in the porosity of the electrode. The selection of a suitable immobilization method depends on the nature of the biological ligand, on the type of electrode used, on the physicochemical properties of the analyte to be detected and on the operating conditions of the electrochemical system. The two methods most commonly used are adsorption and covalent coupling. The physical adsorption of a ligand based on Van Der Waals attractive forces is the oldest and simplest method of immobilization. It does not require any chemical modification of the ligand and makes it possible to regenerate the biosensor or the biofuel cell. The major advantage of this method is its simplicity. However, loss of adsorbed ligand is possible if changes in pH, in ionic strength or in temperature occur during the measurements carried out with the biosensor or the operating of the biofuel cell. Covalent coupling can be used to allow immobilization on a matrix or directly at the surface of an electrode. These methods are based on the reaction between a functional, group of the ligand and reactive groups of the surface of the electrode, most commonly by means of a redox mediator. Indeed, in most biosensors and biofuel cells, the redox centers of enzymes are too far from the surface of the electrode to provide good conductivity of electrons and must be connected to the electrodes by means of redox mediators. The latter play the role of an electron shuttle between the biomolecule and the electrode. The redox mediators may, for example, consist of an electrically conducting linker arm. By way of example, mention may be made of U.S. Pat. No. 5,089,112, which describes a biosensor consisting of a current collector (carbon electrode) connected to a redox enzyme by means of a flexible siloxane comprising a ferrocene group.
One of the difficulties encountered during the production of such biosensors therefore lies in the development of these redox mediators. One of the major issues in this research field therefore consists in managing to directly connect the biomolecules to the surface of the electrodes in order to dispense with the redox mediator.
In order to be able to do without redox mediator, the electrode material must combine several obligatory criteria:                be a conductive material with a large specific surface area, greater in particular than that of conventional carbon fibers,        having an adjustable porosity in order to allow attachment of the enzyme by impregnation and diffusion of the enzyme substrates,        be biospecific for electrochemical reactions in reasonable potential ranges in a biological medium and with respect to biomolecules or entities dissolved in biological media,        be biocompatible.        
Carbon is a material of choice for preparing electrodes. Its chemical inertia in fact makes it possible to explore large ranges of potentials in electrochemistry. For this reason, carbon is very widely used in various forms for preparing electrochemical devices: sensors, actuators, batteries and storage batteries. Furthermore, carbon has the particularity of being a material onto which organic molecules and polymers are effectively adsorbed. It is therefore possible to adsorb thereon redox mediators, enzymes or else conductive polymers for preparing advanced, effective and selective electrochemical devices. Carbon is, in addition, a biocompatible material, which lends itself ideally to the production of devices for biological applications. Carbon has other advantageous properties, which are mechanical strength, thermal stability and an ability to be formed on large scales (discs, films, monoliths of varied shapes).
Carbon electrodes are generally in the form of mesoporous materials. However, the performance levels of the present materials are further limited by currents of which the intensity is too low, by adsorptions which are not very stable or not effective enough and by kinetic limitations due to a low mass transport when the materials are strictly mesoporous. For example, in the case of biofuel cells, the powers generated are insufficient for biomedical devices, such as the supply of electricity to implanted biosensors, in particular.
Increasing the current density of a biosensor or of a biofuel cell is an imperative step for being able to achieve, respectively, sufficient detection limits or powers greater than 2 μW. To do this, it is necessary to increase the specific surface area of the electrodes while at the same time maintaining a sufficient mass transport.
Patent application US 2007/0062821 describes an electrically conducting porous carbonaceous material of which the pores are functionalized with a redox enzyme. The material used in this case consists of a porous metal framework called “metal foam” (metals and metal alloys chosen from Ni, Cu, Ag, Au, Ni/Cr for example), the surface of which is ac least partly covered with a carbonaceous material such as a carbon powder (Ketjenblack in particular), carbon nanotubes or a fullerene. In this type of support, the immobilization of the enzymes can be carried out without using a redox mediator; however, the metals used to produce the framework of this device and the carbon nanotubes covering it are not biocompatible. The productivity of such a device is, moreover, only 4 mA/cm2, which is still not satisfactory.
Materials which are in the form of porous carbon monoliths constitute materials of choice for numerous applications such as water and air purification, adsorption, heterogeneous-phase catalysis, the production of electrodes that can be used as a biosensor or as biofuel cells, and energy storage, owing to their large specific surface area, to their large pore volume, to their insensitivity to the surrounding chemical reactions, to their excellent mechanical properties and, finally, to their biocompatibility.
These materials comprise a high specific surface area and a hierarchical structure, i.e. a cellular structure generally exhibiting a double porosity. They exhibit in particular a mesoporous structure in which the mean pore diameter varies from about 2 to 10 nm.
They can be prepared according to two main families of processes.
The first main family of processes uses soft templates and corresponds to the soft templating methods, i.e. to the methods employing organic/organic interactions between a thermopolymerizable polymer (generally carbon precursor) and certain block copolymers of nonionic polymer type, such as the products sold under the trade names Pluronic® P123 or F127 by the company BASF, which are used as modeling agent in order to directly obtain a porous carbonaceous material after carbonization under an inert atmosphere at 350° C. and pyrolysis (Meng Y. et al., Angew. Chem. Int. Ed., 2005, 44, 2).
The second main family of processes uses rigid templates and corresponds to the hard templating or exotemplating methods, i.e. to the methods in which a mesoporous solid template is impregnated with a solution of a precursor of the final material which it is desired to obtain (carbon precursor, for example), before being carbonized under a nonoxidizing atmosphere.